SUMMARY Serotonin is a neurotransmitter thought to be involved in many behaviors. It is released by a small number of neurons throughout the brain. Little is known, however, about when and where serotonin is released, in the context of well-controlled behaviors in mammals. This proposal aims to determine functions of serotonin- and non-serotonin-releasing neurons in the dorsal raphe, the primary source of forebrain serotonin. The behaviors to be studied involve rewards and punishments, simple motivational stimuli that elicit similar behavioral responses across mammals (including humans). The goal of the project is to link action potentials from identified serotonin-releasing neurons to action potentials in nearby glutamate- or GABA-releasing neurons, and midbrain dopamine-releasing neurons, in the context of motivated behavior. Three aims test three hypothesis that address different mechanistic questions about the functions of serotonin-releasing neurons: 1) dorsal raphe neurons signal reward and punishment rate and timing; 2) dorsal raphe signals reward probability and affective decision bias; 3) dorsal raphe is involved in generating dopaminergic reward activity. Observing activity from specific neuronal types (serotonin-, glutamate-, or GABA- releasing) in the dorsal raphe, and manipulating them while observing activity from dopamine-releasing neurons, while mice perform well-controlled behavioral tasks, will enable testing these three hypotheses. Ultimately, understanding when and where serotonin is released in the brain will be necessary in understanding affective and motivational disorders such as drug addiction and depression.